BRNO UNIVERSITY OF TECHNOLOGY
VYSOKÉ UČENÍ TECHNICKÉ V BRNĚ
FACULTY OF CHEMISTRY
INSTITUTE OF PHYSICAL AND APPLIED CHEMISTRY
FAKULTA CHEMICKÁ
ÚSTAV FYZIKÁLNÍ A SPOTŘEBNÍ CHEMIE
REMEDIATION POTENTIAL OF HUMIC ACIDS
REMEDIAČNÍ POTENCIÁL HUMINOVÝCH KYSELIN
PH.D. THESIS
ZKRÁCENÁ VERZE DIZERTAČNÍ PRÁCE
FIELD OF STUDY: PHYSICAL CHEMISTRY
OBOR: FYZIKÁLNÍ CHEMIE
AUTHOR
Ing. ANNA UHROVÁ
AUTOR PRÁCE
SUPERVISOR
Doc. Ing. Jiří Kučerík, Ph.D.
VEDOUCÍ PRÁCE
1
KEYWORDS
modified humic acids, surface activity, biological activity, soil remediation
KLÍČOVÁ SLOVA
modifikované huminové kyseliny, povrchová aktivita, biologická aktivita, půdní
remediace
MAIN DOCUMENT AVAILABLE AT
Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University
of Technology, Purkyňova 118, 612 00 Brno, Czech Republic
2
TABLE OF CONTENTS
1 INTRODUCTION ................................................................................................... 5
2 LITERATURE REVIEW ........................................................................................ 5
2.1
2.2
Humic substances ................................................................................................................. 5
2.1.1 Humic acids.............................................................................................................. 6
2.1.2 Colloidal properties of HS: aggregation and CMC .................................................. 6
2.1.3 Lignite as a source of HS ......................................................................................... 6
2.1.4 Biological activity of HS .......................................................................................... 7
2.1.5 Environmental significance of HS ........................................................................... 7
Soil ....................................................................................................................................... 8
2.2.1 Soil health and soil quality ....................................................................................... 8
2.2.2 Soil remediation ....................................................................................................... 9
2.2.3 Biochar ................................................................................................................... 10
2.2.4 Soil water repellency .............................................................................................. 10
3 THE GOAL OF THE WORK ............................................................................... 11
4 EXPERIMENTAL PART ..................................................................................... 12
4.1
4.2
4.3
Sample preparation ............................................................................................................ 12
Plant grow experiment ....................................................................................................... 12
4.2.1 Plant growth assessment ........................................................................................ 12
4.2.2 Sugar and protein assessment ................................................................................ 13
Influence on soil ................................................................................................................. 13
4.3.1 Soil respiration ....................................................................................................... 13
4.3.2 Soil water repellency .............................................................................................. 13
5 OVERVIEW OF MAIN RESULTS...................................................................... 14
5.1
5.2
5.3
5.4
5.5
5.6
Physico-chemical properties of modified lignite HAs ....................................................... 14
Fatty acids content ............................................................................................................. 18
Surface activity .................................................................................................................. 19
Biological activity towards higher plants ........................................................................... 22
Remediation potential ........................................................................................................ 26
Statistical analysis .............................................................................................................. 29
6 CONCLUSION ..................................................................................................... 30
7 REFERENCES ...................................................................................................... 32
8 ABSTRACT .......................................................................................................... 41
3
4
1 INTRODUCTION
Soil represents a huge fixed carbon reservoir, which is in permanent contact with
other environmental compartments such as waters and air. Thus, its potential
pollution can directly expand through the surface, ground waters and air [1]. Soil
pollution may be caused by industrial accidents as well as by anthropogenic
activities, and represents a long-term source of environmental contamination. While
the contaminants are bind by weak interactions to soil compartments [2, 3], at any
time, they may be easily released back to the environment and affect the human food
chain [4, 5]. Thereupon, processes of decontamination of polluted soils are largely
desired. The soil remediation technologies, and relatively inexpensive alternative,
the soil washing technologies represent useful tool for transformation and
detoxification of pollutants [5]. For example, bioremediation enables permanent
elimination of pollutants by in situ remediation, however, is limited by the correct
selection of active microbes, the appropriate soil conditions for microbial activity,
recalcitrance of pollutants to biodegradation, and formation of metabolites, which
may be even more toxic than the parent contaminant [6]. Humic substances,
naturally occurring surfactants, are recognized as a possible aid in soil remediation
techniques [5]. For example, the bioavailability of polychlorinated biphenyls (PCB)
and polycyclic aromatic hydrocarbons (PAH) appeared to be increased by addition
of oxygenous humic substances to contaminated soils [7, 8]. Nowadays, one of the
main sources of humic substances represents lignite and peat, both in the past used
mainly as a not very effective fuel in power plants. Lignite represents the youngest
type of coal with the age belonging between peat and brown coal. From the physical
chemistry point of view, lignite can be described as a system with a variable and
unique surface morphology. From the chemical point of view, lignite is a very
heterogeneous composite – except humic substances, also plant residues, bitumens,
mineral inclusion and mainly high content of water can be identified in the structure.
It has been already recognized that the most attractive way of non-energetic
exploitation of lignites is their use as a source of humic substances [9].
2 LITERATURE REVIEW
2.1
Humic substances
Humic substances are the most widely distributed natural products on the earth´s
surface, occurring in soils, lakes, rivers, and the sea [10]. In spite of their extensive
distribution, much remains to be learned about their origin, chemical structure and
reactions. Comprehensive historical review of chemical investigation of humic
substances is referred in the book of Kononova [11]. In 1994 Stevenson [12] stated
that soil organic matter includes a broad spectrum of organic constituents, many of
which have their counterparts in biological tissues. However, to simplify this very
complex system, two groups of organic compounds can be distinguished: nonhumic
substances and humic substances. The classical definitions of HS are based on
solubility properties in the aqueous solutions used as soil extractants. HS are usually
5
divided into three main fraction (1) humic acids (HA), which is soluble in alkaline
solution but is precipitated by acidification of alkaline extract; (2) fulvic acids (FA),
which is that humic fraction which remains in the aqueous acidified solution, i.e. it
is soluble in both acid and base; and (3) the humic fraction that cannot be extracted
by dilute base and acid, which is referred to as humin.
2.1.1 Humic acids
Humic acids represent the fraction of HS that is not soluble in water under acidic
conditions (pH < 2) but is soluble at higher pH values.
2.1.2 Colloidal properties of HS: aggregation and CMC
HS are a major class of naturally occurring organic colloidal particles, which not
only demonstrate colloidal phenomena by themselves, but display a range of
important colloidal interactions in the presence of other substances. Specifically, HS
not only interact with other naturally occurring soil components such as clays and
metals ions, but also with man-made materials such as herbicides and pesticides
used in agriculture [13]. Aqueous solutions of synthetic surfactants have a
characteristic concentration known as the critical micelle concentration (CMC), at
which the monomers spontaneously aggregate to form micellar assemblies. The
same has been reported for the concentrated HA solutions, which have estimated
CMC values as high as 10 g L-1 [14, 15]. In other experiment [16] several HS were
analyzed by DOSY-NMR and the apparent CMC was established to be greater than
4 g L-1. For dilute HA solutions, however, Engebretson et al. [17, 18] found
evidence for micelle-like organizations which does not feature a CMC. In this
model, the amphiphilic HA molecules are considered to "aggregate" both intra- and
intermolecularly. The former is made possible by the chain length and flexibility of
the humic polymer, which allow them to fold and coil in a manner that directs
hydrophilic (e.g. carboxy and hydroxy) groups outward and keeps more
hydrophobic (e.g. hydrocarbon) moieties isolated in the center. This process, which
could in principle occur also with a single polymer strand, produces an entity that is
operationally similar to a conventional micelle, albeit more structurally constrained.
Like a micelle, it has a hydrophobic interior and a more hydrophilic surface, giving
it distinct solubilizing powers for nonpolar solutes. To indicate both similarities and
differences with normal surfactant micelles, these HA structures have been referred
to as pseudomicelles.
2.1.3 Lignite as a source of HS
Lignite in the Czech terminology includes a variety of brown coal, which exhibits
the lowest degree of coalification, is mostly of xylitic character with large or small
fragments of wood and tree trunks with preserved growth rings (the elemental
analysis of particular products of the process of coalification is reported in Table 1.
From the petrological viewpoints, it is a brown coal hemitype. Its calorific value is
lower than 17 MJ/kg. Lignite represents the lowest quality mineral fuel,
6
consumption of which gradually decreases [19]. The largest deposits of lignite in
Czech Republic occur along the northern margin of the Vienna Basin, which extends
from Austria into southern Moravia. Lignites are almost entirely used as fuel.
Moreover, because of low calorific value, lignites were recognized to be low-cost
raw materials suitable for non-fuel utilization. In this case, mainly specific surface
properties of lignite particles were exploited. The important and promising field of
exploitation is in agriculture for increasing of soil fertility and nutrients availability
[20]. In environmental protection industry lignites could serve as materials for
treating or preventing ecological accidents, e.g. in remediation technologies [5]. Due
to their high cation exchange capacity attributed mainly to high content of HS,
lignites can effectively serve as organic components in organo-mineral fertilizers
[21]. Subsequently, addition of lignites to fertilizers can increase the yield and
improve the quality of growing plants and crops.
2.1.4
Biological activity of HS
Increasing evidences have indicated that humic substances can induce plant growth
[22]. This effect has often been attributed to the formation of complexes between HS
and nutrients [23]. However, it was demonstrated by many authors that HS act as
protein carriers of ions trough permeable cell membrane [22, 24], they activate
respiration, the Krebs cycle, photosynthesis and production of adenosine
triphosphate [22, 25, 26]. The mechanism by which HS stimulate plant biological
activity is not still well clarified, however, it is already clear that origin, molecular
size, chemical characteristics, pH and concentration play a crucial role. It was
demonstrated that functional carboxylic and phenolic C groups seem to have an
important role in determining their biological activity [22, 27]. Many authors have
demonstrated that low-molecular-size (LMS) fractions of HS are biologically more
active than the high ones [28, 29]. The LMS may enter the plant and affect plant
metabolism by either enzyme activation or inhibition and inducing or repressing
protein synthesis and functional changes in root architecture [30-32]. More recent
works suggested the hormonal activity of HS [33, 34]. Pinton et al. [35] showed that
low molecular weight water extractable humic fraction affects nitrate uptake and
plasma membrane (PM) H+-ATPase activity in maize roots. This findings was
supported by Canellas et al. [34], who showed that HS isolated from earthworm
compost induced maize H+-ATPase activity, while Zandonadi et al. [36] investigated
the effect of HAs on lateral root development concerted with PM H +-ATPase
activity. This implies that predominantly LMS fraction of HS can serve as an
environmental source of auxin-like activity [33, 34]. (Auxins are hormones which
are involved in plant cell elongation, apical dominance and rooting [36].)
2.1.5
Environmental significance of HS
Humic acids and related substances are among the most widely distributed organic
materials on the Earth. HS are fundamental in geochemistry and in the environment
for the following reasons [12]:
7
They may be involved in the transportation and subsequent concentration of mineral
substances. HS can serve as carriers of organic xenobiotics (as well as trace
elements) in natural waters. HS act as oxidizers or reducing agents, depending on
environmental conditions. The sorption capacity of the soil for a variety of organic
and inorganic gases is strongly influenced by humus. The ability of the soil to
function as “sink” for N and S oxides in the atmosphere may be due in part to
reactions involving organic colloids. HS provide numerous benefits to crop
production. They help break up clay and compacted soils, assist in transferring
micronutrients from the soil to the plant, increase seed germination rate and
penetration, enhance water retention, and stimulate development of micro flora
populations in soils. HS also slow down water evaporation from soils. Their benefits
have been proven both experimentally and in the field [37]. Environmental
significance of HS is in their strong association with organic and inorganic
compounds in soil and water, acting as both storage and transport agents for these
species.
2.2
SOIL
There are many different opinions as to what constitutes soil, and there is no
commonly agreed definition [38]. A more all-embracing is the definition that soil is
a natural body composed of minerals, organic compounds, living organisms, air and
water in interactive combinations produced by physical, chemical and biological
processes. The primary components of soil are inorganic material, mostly produced
by the weathering of bedrock or other parent material [38].
2.2.1
Soil health and soil quality
Soil health is defined as the continued capacity of soil to function as a vital living
system, by recognizing that it contains biological elements that are key to ecosystem
function within land-use boundaries. These functions are able to sustain biological
productivity of soil, maintain the quality of surrounding air and water environments,
as well as promote plant, animal, and human health [39, 40]. Soil quality cannot be
measured directly, but soil properties that are sensitive to changes in management
can be used as indicators [41]. Soil health indicators are needed that help
smallholder farmers understand the chain of cause and effect that links farm
decisions to ultimate productivity and health of plants and animals. The quality of
soil is rather dynamic and can affect the sustainability and productivity of land use.
Identification of biological indicators of soil quality is reported as critically
important by several authors [42, 43] because soil quality is strongly influenced by
microbiological mediated processes (nutrient cycling, nutrient capacity, aggregate
stability). Biological indicators of soil quality that are commonly measured include
soil organic matter, respiration, microbial biomass (total bacteria and fungi,) and
mineralizable nitrogen. Soil organic matter plays a key role in soil function,
determining soil quality, water holding capacity and susceptibility of soil to
degradation [44]. In addition, soil organic matter may serve as a source or sink to
8
atmospheric CO2 and an increase in the soil C content is indicated by a higher
microbial biomass and elevated respiration [44, 45]. It is also the principal reserve of
nutrients such as N in the soil and some tropical soils may contain large quantities of
mineral N in the top 2m depth [44]. Chemical indicators: In order to achieve high
crop yields smallholder farmers have to provide soil nutrients in large quantities
[46]. Therefore it is possible to alter the pool of available nutrients by adding
inorganic fertilizers, incorporating cover crops, and using other organic materials in
form of manures and composts [47]. Results of chemical tests are soil quality
indicators, which provide information on the capacity of soil to supply mineral
nutrients, which is dependent on the soil pH. Soil pH is an estimate of the activity of
hydrogen ions in the soil solution. It is also an indicator of plant available nutrients.
High activity is not desirable and the soil may require liming with base cations Ca or
Mg in order to bring the solution back to neutral. Physical Indicators - soil physical
properties are estimated from the soil’s texture, bulk density (a measure of
compaction), porosity, water-holding capacity [48]. The presence or absence of hard
pans usually presents barriers to rooting depth. These properties are all improved
through additions of organic matter to soils. Therefore, the suitability of soil for
sustaining plant growth and biological activity is a function of its physical properties
(porosity, water holding capacity, structure, and tilth).
2.2.2
Soil remediation
Soil and ground water pollution became a major environmental issue for the
developed world later than, for example, air pollution or waste treatment [49]. In the
1980s the developed world became much more aware of the significance of soil
contamination as an environmental issue. In many countries, research institutes and
companies began to develop technologies to solve the problem of contaminated soil.
Most of these were based on treatment of excavated soil. Thermal desorption and
soil washing were typical technologies resulting from this phase of development.
These technologies, which are often also heavy consumers of energy have become
known as “intensive” treatment technologies [50]. However, these technologies can
be associated with high energy consumption and sometimes lead to new
environmental problems [49]. So, both from an environmental and an economic
point of view, there is an emerging international demand for low input, low energy
remedial technologies. These technologies have become known as “extensive”
technologies. Being low input, extensive technologies may often be long-term
treatments, and so their application implies a more holistic and longer-term
management of the risks from contaminated land than is the case for rapid intensive
response [50].
Due to the fact that HA are defined as a surface active substances, based on their
significant effects on surface tension and with their tendency to complex metal ions
and sequester organic molecules (see chapter 2.1.5), it could be suggested that they
may be used to remove contaminants from polluted water. This has been recognized
9
in various studies [51-54] and is the subject of a patent by Zanin and Boetti, who
reported the use of extracted HA for the removal of heavy metals, chlorinated
organics, phosphorus, and nitrogen from waste water [55]. The main difficulty in
using HA for this purpose, however, is that their isolation from natural matrices
(usually soil) is laborious, time consuming, and costly. One exception to this is
leonardite humic acid (LHA), which is available in bulk and requires little or no
further treatment. LHA is a material found in association with leonardite, lignite
distributed in vast deposits across North America and it presently enjoys wide use as
an agricultural soil conditioner [56]. HA prepared in this work are produced from
lignite as well. Therefore, one can observe some similarities with LHA.
2.2.3
Biochar
One of the most popular soil organic amendments of recent times is biochar. It is
carbonaceous porous material obtained by pyrolysis of biomasses. It shows great
potential in improving soil fertility [57-59]. The effect of biochar on soil biological,
chemical and physical properties is complex but, in general, it establishes a carbon
sink and maintains soil fertility for over the long time of soil cultivation. This was
proved by many authors and agriculturists on many types of used soils. [60-62]
Among others the most advantageous application of biochar is in remediation of
contaminated soils [63, 64]. In the experiment of Abdelhafez et al. 2013 [65], the
restoration of shooting range soils with high lead contamination by application of
biochar was successfully provided. The results showed that the addition of biochar
significantly increased the soil water holding capacity, availability of nutrients (N, P
and K), cation exchange capacity, and stimulated the microbial growth (bacteria and
fungi) in soil. Many studies have indicated that the carbon in biochar remains stable
for millennia, what resulted in reducing of greenhouse gas emission [66]. In
summary, biochar maintain soil fertility and establish a carbon sink, enhances the
microbial and chemical transformation and cause a significant increase in crop
production.
2.2.4
Soil water repellency
Soil water repellency (SWR) is a physical property of soil that limits water
infiltration, as well as a chemical property in it that consists of organic compound. In
a hydrophobic soil, water will not readily penetrate and infiltrate into the soil, but
will blockade and remain on the surface [67, 68]. Surface attraction of repellency for
water originates from the attractive forces between water and solid surfaces. If the
attraction is greater between water and soil particles than between individual water
molecules, the water will spread out and be absorbed. However, when the attraction
between water molecules is greater than that between the water and soil surface, the
water will be repelled by the soil particle rather than infiltrate [67, 68]. The main
factors affecting the soil water repellency are soil texture and organic matter [67].
Hydrophobic substances are naturally occurring and derived from organic
compounds of most living and decomposing plant species or microorganisms. The
10
most commonly associated species with water repellency are those containing
significant amount of resins, waxes and aromatic soils [67-69]. Organic compounds
most suspected to cause SWR among others are aliphatic hydrocarbons and
amphiphilic compounds such as long-chained fatty acids. Fatty acids form very
insoluble soaps with calcium, magnesium and other bi and trivalent metals, when
dry, they become extremely water-repellent [70].
3 THE GOAL OF THE WORK
The main goal of this work is the optimization of procedures leading to the
production of modified lignite humic acids, which have improved properties and
modified chemical characteristics applicable in environmental technologies. In light
of above discussion, this challenge seems to be very ambitious and also very
optimistic. In fact, this study follows previous pilot researches carried out at Faculty
of Chemistry of Brno University of Technology, focused on production of modified
humic acids [71, 72]. In mentioned works, two ways of modification of parent
lignite were chosen, i) chemical and ii) physical. In both cases, humic acids were
extracted by standard procedures [9, 73-75]. It was demonstrated that both types of
modifications provided possibilities to produce humic acids, which have biological
activity comparable with commercially available biostimulators [76]. In fact, the
highest biological activity showed humic acids produced using way “ii)”. In our
previous work [77], using the same set of humic acids, the effect of products on the
surface tension was studied. It was shown that the way ii) is slightly more efficient
to produce HA with higher surface activity than was “i)”. The way “i)” was studied
more deeply in the recent work of David et al. [78] who used a comprehensive
approach in which a multitude of techniques was used to characterize humic
products; however applied statistical approach revealed only weak correlations
between primary and secondary characteristics of HA. Nevertheless the promising
potential of humic acids as a sorbent and biostimulant were confirmed and
discussed. In this work, the production of a set of “modified” humic acids (MHA) is
planned also using approach “ii)”. This approach mimics the processes observed in
rhizosphere where “small” organic molecules (typically acids) are produced by root
system of higher plants and possibly cause the reaggregation of HA present in
rhizosphere [79]. As a result, apparently large molecules of humic acid are
disaggregated into small subunits or even molecules, which allow and support the
transport of nutrients and simultaneously, they are able to penetrate through the root
cell walls and participate in cell biological processes. Therefore, MHA produced in
this work will be characterized mainly for their biological and surface activity. In
other words, the aim of this research is to find out the optimal physical pre-treatment
of raw lignite with respect to maximal surface activity and biological activity.
Furthermore, we would like to continue the search for the relation between the type
of lignite modification agent and the HA structure and properties in order to design
an approach leading to production of humic acids with desired properties.
11
4 EXPERIMENTAL PART
4.1
SAMPLE PREPARATION
Ten modified humic acids (MHA) were prepared by pre-treatment of parental
lignite. To obtain MHA samples, milled and sieved (at 0.2-0.3 mm) lignite was first
pre-treated by various organic acids. Their list is given in Table 2.
Table 2 List of studied samples
Sample
MHA0
MHA1
MHA2
MHA3
MHA4
MHA5
MHA6
MHA7
MHA8
MHA9
MHA10
4.2
Modifier
None
formic acid (20%)
acetic acid (20%)
propionic acid (20%)
butyric acid (20%)
maleic acid (20%)
benzoic acid (0.3%)
fumaric acid (0.5%)
oxalic acid (10%)
phenylacetic acid (1.5%)
picric acid (1.4%)
PLANT GROW EXPERIMENT
To determine biological activity of extracted HK the experiment with the maize
growing hydroponically in HA solutions was carried out. For the experiment, the
corns of the common maize Zea mays CEKLAD 235 species (Oseva Bzenec Ltd.,
Czech Republic) were used. The species was chosen for its wide spread of use for
kernel and silage purpose, high percent of germinative activity and high durability.
The experiment was carried out according to previous work of [36, 78, 80].
4.2.1
Plant growth assessment
All 30 sprouts and later the grown seedlings for each sample solution were weighted
and the difference was calculated as a total mass increment. The roots of five
selected seedlings were measured before and after the growing experiment and the
difference was calculated as a root length increment. The roots of previously
selected five seedlings were scanned against black paper background using common
desktop office scanner. The obtained images (300×300 dpi, 2424×3426 pix and 24
bits pix–1) were loaded by Harmonic and Fractal image Analyzer software (HarFa)
(http://www.fch.vutbr.cz/lectures/imagesci) [81], trimmed to desired square images
(300×300 dpi; 2048×2048 pix×24 bits pix–1), saved as bitmaps and subjected to 2D
Wavelet Analysis with black and white thresholding set at the value of 160. The
12
K[BW] value represents the number of pixels on the black and white border,
therefore the value stands for the measure of root division [78, 81, 82].
4.2.2
Sugar and protein assessment
After the plant grow experiment, total dry material was subjected to sugar and
protein assessment. The sugar content was determined polarimetrically using the
customary Ewers polarimetric method [83]. The proteins were determined by the
Kjeldahl method [84] for the determination of total nitrogen. The measurement was
performed in cooperation with Mendel University Brno.
4.3
INFLUENCE ON SOIL
To assess the soil remediation potential of studied HAs samples, common soil
experiments such as soil respiration and soil hydrophobicity were performed.
4.3.1
Soil respiration
Soil respiration is one of the main soil quality indicators and its measurement
represents a common tool to evaluate soil biological processes. It reflects the
primary path by which CO2 returns to the atmosphere [85-87]. In such experiments,
biological activity is usually assessed by the measurement of CO 2 evolution, O2
consumption, microbial activity, enzyme activity, or other parameters. In present
work, the soil respiration experiment was carried out with the aim to prove the
positive remediation effect of lignite humic acids on soil microbiological activity.
Incubation experiment was carried out with RESPICOND device by measuring of
electrical conductivity of the KOH solution suspended above the vessel with studied
soil to monitor the CO2 evolution during the experiment in varying intervals from 30
to 60 minutes. The soil respiration measurements were carried out in three
replicates, which are presented in this study as averaged values. Measurement of
evolved CO2 began immediately within 2 h after re-moistening and ended after 50
days of incubation at a constant temperature 20 °C [85]. Data obtained from soil
incubation experiment were fitted by a two-compartment (double-exponential)
model function (using Origin 7.5) [88] Ct = Cr exp(–kr tr) + Cs exp(–ks ts), where Ct
is total carbon in the soil at time t, Cr is the rapidly mineralizable C pool, Cs is the
slowly mineralizable C pool, kr and ks represent the mineralization constants and tr
and ts are respective time constants [88].
4.3.2
Soil water repellency
To assess the changes of wettability / hydrophobicity of the soils treated by studied
humic acid samples before and after incubation experiment, the soil water repellency
experiment (SWR) was carried out [89, 90]. The SWR for all studied samples was
determined by measuring of contact angle by Wilhelmy plate method. One
measurement was provided before and one after incubation experiment on the soils
treated with studied MHA samples. The principal of this method was described in
the textbook of Butt et al. [91]. A thin plate of glass, platinum, or filter paper is
13
vertically placed halfway into the liquid. Close to the three-phase contact line the
liquid surface is oriented almost vertically (provided the contact angle is 0°). Thus
the surface tension can exert a downward force. One measures the force required to
prevent the plate from being drawn into the liquid. After subtracting the
gravitational force this force is 2lγ, where l is the length of the plate. The adaption of
Wilhelmy plate method to soil science was described in detail in the paper of Diehl
[89] and Diehl et. al [90].
5 OVERVIEW OF MAIN RESULTS
5.1
PHYSICO-CHEMICAL PROPERTIES OF MODIFIED LIGNITE HAS
Molecular size distribution
In order to assess the changes in physical structure and self-assembling behaviour of
HA induced by modification of raw lignite, the molecular-size distribution was
analysed. High performance size exclusion chromatography (HPSEC) was carried
out, because it is recognized as a highly precise method for evaluation of the relative
molecular-size distribution of dissolved HAs [92-95].
Figure 1 Comparison of HPSEC chromatograms obtained from UV (280 nm) and
RI detectors of not modified HA and HA treated with formic acid and propionic
acid.
The HPSEC records were obtained using two detectors; UV detector set at 280 nm,
which could detect only chromophores (i.e. predominantly unsaturated moieties),
and refractive index (RI) detector, monitoring elution of whole sample mass. Both
14
detectors recorded similar shape of chromatograms consisting of two peaks
(Figure 1). The first peak was eluted at retention time 23 min and indicated the
elution of the largest components (aggregates) of HAs samples. Conte et al. [96]
demonstrated that this fraction with the largest molecular size is rich in aromatic,
aliphatic and alkyl components. Aliphatic structures contained preferentially longer
alkyl chains, which strongly associated due to stronger hydrophobic effect and
formed supramolecular structures, which eluted at short elution times typical for
apparently large molecular-size components. Shorter alkyl chains than associated in
lower molecular-size fractions eluted with increasing elution times, represented by
the second broad peak with maximum at approximately 32 min. The elution of
aromatic structures decreased with reducing molecular size and increasing elution
time [96]. While the least intensive first peak, and thus the lowest amount of large
aggregates excluded, was recorded for HA treated with formic acid, the most intense
first peak was recorded for HA treated with propionic acid. This observation was
completed by the results from weight-average molecular weight (Mw) calculations,
while both detectors indicated an increase in Mw, except formic acid, comparing to
non-treated HA. The Mw was calculated based on respective calibration from both
detectors and gained values for all samples are listed in Table 6.
Table 6 List of weight-average molecular weight in g mol-1 obtained from HPSEC
analysis (UV and RI detector) of all studied MHA samples.
Sample
Modifier
MHA0
MHA1
MHA2
MHA3
MHA4
MHA5
MHA6
MHA7
MHA8
MHA9
MHA10
none
formic a.
acetic a.
propionic a.
butyric a.
maleic a.
benzoic a.
fumaric a.
oxalic a.
phenylac. a.
picric a.
Mw (g mol–1)
UV
RI
10060
29330
8373
27780
13480
38590
14650
38640
10110
29520
11810
32900
13010
30140
11730
33700
13210
35830
16940
46900
10750
31160
While the lowest Mw was obtained for formic acid, the highest values showed HA
treated with phenylacetic acid ˃ propionic acid ˃ acetic acid (Table 6). This could
indicate the fact, that during treatment, only very small organic acid molecules (i.e.
formic acid) could diffuse more easily through loosely-bound HA aggregates in
lignite and could cause the rearrangement of larger compartments into the smaller
aggregates [93, 97], while the other organic acids act in a different way. Kučerík et
al. [9] showed that addition of formic acid into the HAs solution, caused the
15
protonation of the functional group with subsequent formation of intermolecular Hbonds, leading to larger Mw in comparison with propionic acid. However, the
propionic acid still showed lower Mw value than parental HAs [9]. It indicates that
behaviour of already extracted HAs differs in comparison with HAs still present in
lignites. This might be caused by interactions of humic acids with other lignite
compartments, which might be both weak interaction and covalent bonds. The latter
can be disrupted during extraction as suggested by other studies [98]. This, however,
also implies that, at least for lignites, the results do not support the supramolecular
theory suggested by Piccolo [79] and support the alternative explanation considering
humic acids as “molecular artefacts” [98] produced and largely influenced by used
extraction technique.
Table 7 Molecular size distribution calculated from data detected by UV (280 nm)
detector.
Sample
Modifier
MHA0
MHA1
MHA2
MHA3
MHA4
MHA5
MHA6
MHA7
MHA8
MHA9
MHA10
none
formic a.
acetic a.
propionic a.
butyric a.
maleic a.
benzoic a.
fumaric a.
oxalic a.
phenylac. a.
picric a.
Fraction of molecules (%) in the molecular mass
interval (kDa) based on VWD-UV (280 nm)
0-25
25-50
50-75 75-100 100-150 ˃150
40.8
19.5
14.7
14.5
10.6
0
46.0
16.7
11.9
13.0
12.5
0
30.3
16.2
13.8
16.8
23.0
0
24.0
13.2
12.9
18.9
31.0
0
39.4
17.6
13.2
14.6
15.2
0.1
34.1
16.9
13.7
16.3
19.1
0
31.9
16.8
14.0
17.4
20.0
0
35.2
16.8
13.4
16.1
18.5
0.1
30.3
16.0
13.9
17.8
22.1
0
24.7
14.9
13.4
18.1
28.7
0.3
38.7
16.6
12.4
14.6
17.7
0
To study more in detail the differences in distribution of molecular size after the
modification of parental lignite among individual HA samples, the area under the
peaks was divided into six intervals of Mw, which were divided by total-peak area
and multiplied by factor 100 to obtain percentage contents of particular molecular
fractions. The molecular weights, which were equal or over 150 kDa, were covered
up in one group. Based on UV detector, the most remarkable part of humic
aggregates (24-46 %) occurred in the lowest defined interval of Mw 0-25 kDa. The
most obvious increase was occurred after addition of formic acid into the HS system
(up to 5.2%). On the contrary, addition of propionic acid into the HS system caused
apparent decrease in the formation of low molecular-size aggregates, down to
16.8%. From 12.6 % (formic acid) up to 31.0 % (propionic acid) of HA aggregates
occurred in the interval of Mw 100-150 kDa (UV det.) with only 10.6 % occurred in
non-treated HA. The calculations from RI detector are comparable and confirmative
16
to that obtained from UV detector. The results are summarized in the Table 7 and
Table 8 and illustrated in the Figure 15 and Figure 16, for UV and RI detector,
respectively. Again, the results are in contrast to HPSEC studies reported earlier [9,
79] and underline the importance of extraction procedure.
Table 8 Molecular size distribution calculated from data detected by RI detector.
Sample
Modifier
MHA0
MHA1
MHA2
MHA3
MHA4
MHA5
MHA6
MHA7
MHA8
MHA9
MHA10
none
formic a.
acetic a.
propionic a.
butyric a.
maleic a.
benzoic a.
fumaric a.
oxalic a.
phenylac. a.
picric a.
Fraction of molecules (%) in the molecular mass
interval (kDa) based on RID
0-25
25-50
50-75 75-100 100-150 ˃150
21.1
15.1
10.8
8.6
13.7
30.7
23.3
16.1
10.8
8.4
12.8
28.6
15.5
12.0
8.7
7.1
12.1
44.6
14.4
9.4
6.9
5.9
10.9
52.5
20.4
14.7
10.2
8.1
12.7
33.9
18.5
13.5
9.2
7.4
12.0
39.4
18.3
13.5
9.7
8.0
13.0
37.5
18.5
14.0
9.6
7.6
12.3
38.0
16.7
12.1
8.3
6.8
11.7
44.4
12.4
10.3
7.6
6.4
11.4
51.9
20.7
14.9
9.5
7.2
11.2
36.5
Figure 15 Molecular size distribution calculated from data detected by UV
detector.
17
Figure 16 Molecular size distribution calculated from data detected by RI detector.
5.2
FATTY ACIDS CONTENT
It has already been published by many authors that long-chained fatty acids (FAs)
are one of the groups of compounds responsible for water repellency of the soil [70,
99-101]. Based on their natural hydrophobic tail, Holmberg (2001) [102] classified
FAs as natural surfactants. In the light of the goal of present work, it was convenient
to analyze the content of FAs in individual HA samples, with the main aim, to study
the changes in primary structure induced by modification of parental lignite. FAs
usually occur in the HS in a “free” form and tightly trapped with the humic acid
organic network [103, 104]. The content of FAs in studied HAs samples was
analysed by thermochemolysis, method of chemical degradation, using
tetramethylammonium hydroxide (TMAH). This method allows both,
transesterification of esters and methylation of free carboxylic groups, but does not
distinguish between individual forms of FAs occurring in HS [103]. FAs as methyl
esters (FAMEs), released after thermochemolysis with TMAH, presented in studied
HAs, mostly ranged from the C16 to C32 dominated by the C28 (moltanic acid) and
C30 (melissic acid) members, which are the result of the alteration of biopolymers
such as suberins or waxes of higher plants that underwent depolymerisation (leading
to fatty acids) [105]. Figure 17 shows the distribution of analysed FAMEs in studied
samples. The most FAMEs-rich sample was HA treated with propionic acid. This
indicates that propionic acid is most effective pre-treatment for extraction of fatty
acids, while the other procedures, for unknown reason, decrease the yield
significantly.
18
Figure 17 Content of fatty acids in studied HAs samples.
5.3
SURFACE ACTIVITY
In order to use HAs in environmental applications, such as soil remediation
technologies, one essential condition, i.e. surface activity, must be fulfilled. The
surface activity of HA have already been studied extensively by many authors [56,
106-111]. The surface tension was measured as a function of humate´s water
solution concentration and time. In fact, with increasing concentration the surface
tension progressively decreased. In contrast to our previous research [77], where one
12 hours long measurement was performed, in this work we measured ST of “fresh
surface” produced by an intensive solution shaking and after 24 hours of surface
“aging”. Figure 18a shows the decreasing tendency of ST at both t = 0 h and t = 24
h. and Figure 18b shows their differences. The pattern showed in Figures 18 was
recorded for all studied samples. This behaviour confirms the above mentioned
results from previous studies, that aggregation of HAs molecules takes place from
very low concentrations [112]. The most significant difference (12 mN m-1) in ST
before and after 24-hours repose was observed in HA treated with butyric acid at
concentration 2 g L-1. In overall results and in comparison with not-modified HA,
the most significant decrease in ST was observed in following order: butyric
acid ˃ picric acid ˃ phenylacetic acid. The lowest decrease was observed in case of
propionic acid in all measured concentrations. Generally saying, modifications of
raw lignite carried out in this research induced changes in HAs structure, which
resulted in an increase of its surface activity in aqueous solutions.
19
Figure 18 Surface tension of MHA water solution as a function of concentration at
t = 0h and t = 24h (a). The difference between surface tension at t = 24h and at
t = 0h (b).
Data obtained from ST measurement were subtracted from the surface tension of the
solvent (water) and the results were plotted versus respective concentrations. Plotted
data were fitted by Szyszkowski equation [113], which was generally derived for
water solutions of fatty acids and aliphatic alcohols. With the presumption, that the
main amphiphilic-like molecules occurred in HAs structures are predominantly
aliphatic moieties, such as lipids and alcohols, which are very likely to be excluded
from the bulk of water solution to the surface, the Szyszkowski equation could be
used for fitting. This approach was applied and repeated according to the work of
Čtvrtníčková et. al [77]. Obtained parameters from Szyszkowski eq. are listed in
Table 9. Parameter a reflects the nature of surface active substances and has a
constant value for the surface active moieties of one molecular type, homologous
series. At the time 0 h the parameter a ranged between 4.1 and 14.7 mN m-1
(maximal standard deviation was ± 67 %) and after 24 hours of equilibration
between 2.5 and 6.3 mN m-1 (maximal standard deviation was ± 31 %). While the
20
values at t = 0 h were higher and variable, the values after the equilibration showed
much more similar values. The studied HAs samples can be divided into four groups
according to obtained similar values of parameter a relating to more or less the same
type of molecules absorbed at the surface. To the first group belongs HAs treated
with butyric and maleic acid with the a-parameter between 2.5-2.7 mN m-1, to the
second group not-treated HA, acetic and benzoic acid, to the third group belongs
formic, oxalic and picric acid and the fourth group includes propionic, fumaric and
phenylacetic acid (Table 9). Even if the SD is high (data not shown), the results
show a trend indicating existence of specific groups of molecules at the water/air
interface. Parameter b is different for different molecules and characterizes the
efficiency of surface activity of the absorbed molecules to decrease surface tension
of water [113, 114].
Table 9 Parameters a and b and Гmax (maximal surface saturation) obtained from the
Szyszkowski equation [113] at t = 0h and t = 24h.
t=0h
Sample
MHA0
MHA1
MHA2
MHA3
MHA4
MHA5
MHA6
MHA7
MHA8
MHA9
MHA10
t = 24 h
a
b
a
b
-1
3 -1
-1
(mN m ) (dm g ) (mN m ) (dm3 g-1)
none
7.1
1.6
3.9
46.1
formic a.
8.1
1.2
4.3
35.8
acetic a.
4.1
16.4
3.7
82.7
propionic a.
14.7
0.3
5.6
6.4
butyric a.
9.0
1.0
2.5
2058.8
maleic a.
10.9
0.5
2.7
300.8
benzoic a.
11.0
0.6
3.9
70.2
fumaric a.
11.6
0.5
5.1
18.3
oxalic a.
10.3
0.4
4.0
32.1
phenylac. a.
10.1
0.5
6.3
6.3
picric a.
5.5
3.8
4.5
48.1
t=0h
Modifier
(×10
1.2
1.4
0.7
2.6
1.6
1.9
1.9
2.0
1.8
1.8
1.0
t = 24 h
Гmax
mol m-2)
0.7
0.8
0.6
1.0
0.4
0.5
0.7
0.9
0.7
1.1
0.8
-3
The variability of b-parameter indicated different way of rearrangement, depending
on type of modification agent (Table 9). While in comparison with not-modified HA
(46.1 dm3 g-1), five studied modifiers showed higher efficiency of surface activity
with calculated values ranging between 48.1 (picric acid) and 2058.8 dm3 g-1
(butyric acid), other five modifiers showed lower efficiency ranging from the lowest
values in order phenylacetic acid ˂ propionic acid ˂ fumaric acid ˂ oxalic acid ˂
formic acid (Table 9). For assessment of the surface saturation, the parameter
describing the maximal surface saturation (Гmax) was calculated. After 24 hours of
equilibrating, decreasing tendency was observed for all studied samples (Table 9),
while the highest value was recorded for phenylacetic and propionic acid.
21
5.4
BIOLOGICAL ACTIVITY TOWARDS HIGHER PLANTS
The aim of this part was to assess the biological activity of studied humic acid
samples. The most important parameter in such investigation was the effect on
growth of plants root represented by maize corn. The assessment of biological
activity included measuring of root length increment and weighting of mass
increment of maize plants after the plant grow experiment. The roots length and
weight of five selected seedlings were measured before and after the growing
experiments and the difference was calculated as a root length increment. Since the
experiments were conducted twice, this gave the data for ten seedlings, which were
averaged and the standard deviations were calculated in MS Excel software. Figure
20 shows average root length increment for selected plants growing in solutions of
individual HA samples (results for MHA7 not shown) and for control solution
without any addition of humic acid. As a reference were tested commercial humate
preparations Lignohumate B and Lignohumate AM and as a control was measured
solution of CaCl2. It was evident that addition of humic acids had positive effect on
the root growth increment. The most significant increase in root length was obtained
for HA treated with oxalic acid, non-treated HA and HA treated with propionic acid.
On the contrary, the smallest root length increment was calculated for control
solution and for HA treated with butyric acid. To assess the total mass increment
during the growing experiment, all 30 sprouts and later the grown seedlings for each
sample solution were weighted and subtracted. Figure 21 shows average total mass
increment for all maize plants growing in solutions of individual HA samples and
for control solution without addition of humic acid. The highest difference was
obtained for HA treated with butyric acid and the lowest difference was calculated
for HA treated with acetic acid (Figure 21).
Figure 19 Diversity of selected root scans of Zea mays grown in MHAs solutions.
The plant root system consists of the primary roots (already assessed by the
measuring of length increment) and secondary roots, the lateral roots. The lateral
roots extend horizontally from the primary root and, while they spread through
rhizosphere, they facilitate the water and nutrient uptake for the growth and
development of the plant. Therefore, their assessment was, together with length and
weight of roots, one of the further main objectives of this part. For the quick and
22
more accurate assessment of root division of lateral roots of maize plants after plant
grow experiment was used HarFa image analyzer software, calculated as a root
division parameter. Image analysis represents useful tool in many field of science.
The most popular is in biological and environmental science [82, 115]. Selected five
roots for each HA-solution were scanned and obtained images (Figure 19) were
subjected to thresholding from the gray scale to black and white images. Obtained
values after tresholding - fractal measure K[BW] - showed the number of pixels on
the black and white border what corresponded to the root division. With increasing
value of K[BW] pixels, the longer is the border between root and background and
bigger is the growth increment. Figure 22 shows average root division of five
selected maize for each studied HA sample. The highest root division was observed
for HA treated with butyric acid.
Table 10 Biological activity results from plant grow experiments for studied MHA
samples.
Root
length
Mass
Sample
Modifier
increment increment
(cm)
(g)
control (CaCl2) 14
30
MHA0
none
20
34
MHA1
formic a.
17
31
MHA2
acetic a.
18
27
MHA3
propionic a.
19
32
MHA4
butyric a.
16
36
MHA5
maleic a.
17
33
MHA6
benzoic a.
19
32
MHA8
oxalic a.
21
33
MHA9
phenylac. a.
18
29
MHA10
picric a.
16
32
lignohum B
16
36
lignohum AM 17
28
Root
division
K[BW]
(pix)
34936
73317
33705
36855
27374
48800
34415
37065
28817
36070
37712
28061
42962
Starch
content
(%wt)
37
33
35
33
33
27
34
33
34
33
34
34
33
Protein
content
(%wt)
11
11
11
11
11
12
11
12
12
12
13
12
13
Conc. = Concentration
Next parameter of HAs-biological activity was their effect on the starch and protein
content in maize plants after the plant grow experiment. The starch and protein
content assessments were evaluated for 30 whole (but dried) plants and the contents
are shown in Figure 23. Generally, the content of starch varied between 26.6 and
36.9 % (wt). The most remarkable was lower content of starch in the plant growing
in the solution of HA treated with butyric acid. The protein content varied between
10.6 and 12.7 % (wt) with highest content recorded for picric acid. All the results
23
obtained from plant grow experiment are summarized in Table 10 and demonstrate
that all studied samples showed biological activity.
Figure 20 Averaged root length increment of selected Zea Mays plants.
Figure 21 Average total mass increment of all Zea Mays plants.
24
Figure 22 Average root division of selected Zea Mays plants.
Figure 23 Starch (dark grey) and protein (light grey) content in wt%
analysed in the dry mass of Zea mays plants after the plant grow experiment
for studied HA samples.
25
5.5
REMEDIATION POTENTIAL
Currently, application of carbonaceous amendments for soil remediation is very
popular and increasingly utilized by the agriculturists for the reconstruction and
recovery of soil organic matter. However, there is a risk that when extraneous
organic matter is incorporated into the soil, it can cause negative effects on soil
properties such as e.g. water repellency or soil texture. One of the confirmative
examples is uncontrolled disposal of olive mill waste water, when both positive and
negative effects on soil quality have been reported [116]. The aim of this part was to
test the use of humic acids as an organic amendment/remediation agent of soil
damaged by long-term agricultural activity.
Figure 24 Soil respiration and its derivative - respiration rate.
To assess the effect of studied lignite HAs samples as an organic amendment in the
soil, the respiration experiment, as a common tool for evaluation of biological
processes performing in the soils, was carried out. The biological activity of HAs in
soil was assessed by the measurement of CO2 evolution. CO2 is a natural product of
activity of soil organisms and thus its evolution is a positive indication of soil
(microbial) activity [85, 117]. The laboratory soil respiration experiment performed
in this study was carried out under constant optimal moisture condition (76% of pF
1.8) and constant temperature (20 °C) for 50 days. Figure 24 shows the mean value
of cumulative evolved CO2 and the rate of CO2 evolution from five representative
HAs samples (not-treated HA, HA treated with formic acid, acetic acid, propionic
acid and phenylacetic acid). As a reference the pure soil without any treatment
(Control) and soil treated with common chemical detergents such as SDS and
26
Triton X-100, respectively, were analysed. With increasing incubation time, the
respiration rate gradually decreased. The highest respiration rate (0.06 mg CO 2 h-1/
100 g soil) was recorded at the beginning of the incubation experiment immediately
after re-moistening of the air-dried samples. During the first 15 days of the
incubation experiment, the tendency of the CO2 emission rate was rapidly
decreasing (Figure 24). The reasons of rapid emission of CO2 could be attributed to
the anomaly called “Birch effect” [118]; when soils become dry, e.g. during summer
because of lack of rain and are then rewetted e.g. by precipitation or irrigation, there
is a burst of decomposition, mineralization and release of inorganic nitrogen and
CO2 [119], [120],[118]. After the first 15 days, slower and almost constant rate of
CO2 emission was recorded and this tendency persists until the end of the
experiment. After 50 days, the emission decreased to 0.01 mg CO 2 h-1/100 g soil.
This pattern was observed for all studied samples.
Table 11 shows the amount of released CO2 after 50 days of respiration experiment
for all analysed HAs samples. The greatest release of CO2 was observed from the
Control sample, the pure soil without any treatment (24.0 mg CO2 /100 g soil), while
the lowest release was observed from soil mixed up with not-treated HA (18.4 mg
CO2 /100 g soil). Other significant decrease was observed in case of HA treated with
acetic acid (18.3 mg CO2 /100 g soil) but that could be attributed to present lower C
content after the extraction process (see chapter Chyba! Nenalezen zdroj odkazů.).
Generally, all analysed samples showed decreasing tendency of CO2 emission after
50 days of incubation, compared to pure soil, what could indicate both, nontoxicity
of studied HA while in contact with soil and successive stimulation of the soil
connected with gradual release of C, respectively.
Table 11 Released CO2 / 100 g soil after 50 days of incubation experiment.
Parameters Cr, Cs and tr, ts obtained from fitting the model function [88]
Sample
MHA0
MHA1
MHA2
MHA3
MHA9
SDS
Triton
Control
mg CO2 /100 g
Cr
tr
Cs
ts
Cr / Cs
after 50 days (% of Ct) (days) (% of Ct) (days)
none
18.4
18
5
82
54
0.2
formic a.
20.6
19
6
81
53
0.2
acetic a.
18.3
20
6
80
61
0.3
propionic a.
21.9
27
10
73
78
0.4
phenylac. a.
22.3
19
6
81
54
0.2
19.9
16
7
84
120
0.2
21.9
20
6
80
50
0.3
24.0
24
5
76
44
0.3
Modifier
In order to describe the mineralization of total soil organic carbon during the
incubation experiment, data obtained from incubation experiment were fitted by a
model two-compartment (double-exponential) function Ct = Cr exp(–kr tr) + Cs exp(–
27
ks ts) [88], where Ct describes the total organic C consisting of two fractions, the
rapidly mineralizable C pool (Cr) and the slowly mineralizable C pool (Cs). Table 11
shows the calculations of both organic C pools and their respective time constants
(tr, ts). [88, 121] After the modification of parental lignite, the content of rapidly
mineralizable C decreased, while the content of slowly mineralizable C increased.
This was determined for all studied HA samples, except HA treated with propionic
acid, which showed contradictory process. These findings were confirmed by
additional calculation of Cr / Cs ratio (Table 11). The highest Cr was confirmed for
propionic acid (27%) and Control sample (24%), while the lowest Cr was calculated
for SDS (16%) and not-treated HA (18%). Soil incubation experiment carried out in
this research agreed with previously mentioned studies [66] that after addition of
appropriate soil organic amendment into the soil the content of labile C decreases
while the content of stabile C increases. This and previously mentioned behaviour of
studied HAs remarkably remains the behaviour of biochar.
100
before incubation (0.1 g/L)
after incubation (0.1 g/L)
before incubation (8 g/L)
after incubation (8 g/L)
90
Contact angle (°)
80
70
60
50
40
30
Sample
Figure 25 Contact angles before and after the incubation of the soils treated with
selected HAs samples at concentration 0.1 and 8 g L-1, synthetic surfactants (SDS,
Triton X-100) bellow and above CMC, and pure soil (Control).
When the studied HAs samples were mixed up with soil to perform the soil
incubation experiment, the soil water repellency (SWR), as a parameter of soil
wettability, was determined. To study the effect of present HA on the soil
wettability, the SWR was measured both, before and after the soil incubation
experiment. The SWR was determined by measuring of contact angle by Wilhelmy
28
plate method [89-91]. Figure 25 illustrates the results of contact angles recorded for
selected studied HAs samples at two analysed concentrations, 0.1 g L-1 and 8 g L-1,
respectively. As a control, pure soil without any treatment was investigated. For
comparison, common synthetic surfactants, such as SDS and Triton X-100, were
analysed at concentrations bellow and above CMC, respectively. Before incubation
experiment, with increasing concentration increasing tendency of contact angle were
observed for all studied HAs. The extremely high values were found for HA treated
with propionic acid, at both concentrations. On the contrary, after the incubation
experiment, all studied samples showed stabilized values. In summary, before
incubation experiment, only HA treated with propionic acid induced increase in soil
water repellency, all other studied samples showed averaged stabilized values, in
comparison to pure control soil. After the incubation, no significant variations in the
soil-hydrophobocity were observed.
5.6
STATISTICAL ANALYSIS
In this part, the attempt is paid to find the connection between chemical
characteristics of studied HAs samples, their physicochemical properties and effects
on soils and maize. To study their relationship, the Pearson´s correlation coefficient
(r) (MS Excell) was applied. The results are summarized in Table 13. Relationship
between primary and secondary structure of studied HAs was studied by the
correlation between fatty acids and intervals of molecular size of HAs. The positive
linear correlations found between longer-chained FAs (C24-C32) and intervals of
large molecular size 150-200 kDa (r = 0.63) obtained from RI-detector, was in
agreement with the work of Conte et al. [93], who demonstrated that the large
molecular size fractions of HAs are rich in aliphatic structures containing
preferentially longer alkyl chains. No significant correlation found for intervals of
small molecular size 25-50 kDa only confirmed later suggestion. The correlation
coefficients between all FAs and respective intervals of molecular size are shown in
Table 13. Table 13 shows a connection between FAs and ST for concentration
0.5 g L-1. In fact, lower measured concentration 0.1 g L-1 gave no correlation, while
next higher concentration gave still significant, but lower correlation than 0.5 g L-1.
This observation underlines the changing character of humic aggregates in interface
layer at different concentrations. Furthermore, it roughly correlates with observation
of other authors [122-124] that conformation of dilute humic acids changes at
1 g L-1. On the contrary, low correlation coefficients were found between FAs and
parameters a and b, which can be attributed to above mentioned changes in
aggregation. High positive linear correlations (r = 0.65-0.79) were found between
long-chained FAs and parameter of soil hydrophobicity, the contact angle (CA).
This correlation supports the assumption that long-chained FAs are responsible for
soil water repellency [70, 101]. However, the correlations were found only before
incubation experiment (Table 13). It implies that soil microbiological activity
affected the long-chained FAs and they were relatively quickly transformed by soil
microorganisms. We speculate that they were either mineralized or transformed into
29
soil matrix and they were not involved any more in soil hydrophobicity. The
relationship between primary structure of HAs and their biological activity was
demonstrated by relatively high positive linear correlation (r = 0.78) between fatty
acids and parameter of biological activity towards higher plants, the root division
(K[BW]) of maize (Table 13).
Table 13 The Pearson´s correlation coefficients between FAs and parameters of
primary and secondary structure of studied HAs samples and parameters of soil
properties.
25-50 kDa
150-200 kDa
ST (0.1 g L-1)*
ST (0.5 g L-1)*
ST (2 g L-1)*
a-parameter
b-parameter
CA before inc.
CA after inc.
Cr
Cs
K[BW]
C16
0.02
0.43
-0.15
0.40
0.48
-0.11
0.18
0.27
0.34
-0.04
0.35
0.62
C18
0.19
0.25
0.17
0.57
0.34
-0.07
-0.03
-0.01
0.24
0.25
0.61
0.75
C20
-0.30
0.52
0.35
0.74
0.50
0.29
-0.19
0.36
0.63
-0.21
-0.03
0.51
C22
0.12
0.36
0.27
0.70
0.37
0.04
-0.19
-0.01
0.41
0.24
0.53
0.78
Fatty acids
C24
C26
-0.48 -0.50
0.63 0.61
0.12 0.12
0.67 0.67
0.42 0.38
0.46 0.50
-0.35 -0.35
0.79 0.77
0.31 0.38
-0.71 -0.71
-0.43 -0.52
0.13 0.12
C28
-0.48
0.63
0.08
0.65
0.36
0.49
-0.35
0.69
0.48
-0.62
-0.46
0.19
C30
-0.44
0.63
0.06
0.65
0.36
0.45
-0.34
0.62
0.54
-0.53
-0.37
0.25
C32
-0.38
0.63
0.14
0.64
0.44
0.35
-0.32
0.65
0.36
-0.50
-0.16
0.33
*t = 24h
6 CONCLUSION
In this work, a set of ten “modified” humic acids (MHA) was produced using
physico-chemical modification of parental lignite. The multiple methods were used
to characterize the samples and determine the biological and surface activity of
MHAs and its relationship.
The results from elemental analysis and FTIR spectroscopy showed an increase in
aromatic moieties after the lignite pre-treatment. The result from HPSEC analysis
indicated an increase in weight-average molecular weight (Mw) in all studied
samples, only except formic acid. These results were not in agreement with our
hypothesis that small organic molecules could cause the rearrangement of larger
compartments into the smaller aggregates. This was confirmed only for very small
organic acids (i.e. formic acid); the other organic acids acted in a different way.
However, even if the smaller aggregates were produced, they could be simply
washed out during the extraction procedure of HA. Molecular size distribution
showed that in produced MHAs were present mainly the fractions of
molecules/aggregates 0-25 kDa and ˃150 kDa.
30
Fatty acids presented in studied HAs ranged from the C16 to C32, the dominant
fractions were the C28 (moltanic acid) and C30 (melissic acid) members. The
distribution of presented FAs varied with different modifiers, while the richest
sample was HA treated with propionic acid.
Surface tension measurement showed high surface activity of all studied MHAs
samples comparable with chemical surfactants, while the most effective was HA
treated with butyric acid. HA treated with propionic acid showed the lowest
efficiency.
The results obtained from plant grow experiment demonstrated that all studied
MHAs samples showed biological activity and they are able to participate in plant
growth processes.
Soil incubation experiment and water repellency experiment proved that the samples
can be used for soil remediations with no risk of toxicity or initiation of soil
processes leading to an increase in soil hydrophobicity.
The result from statistical analysis showed that the biological activity of HAs was
influenced by their physico-chemical properties. However, the surface properties did
not show any correlations with physico-chemical properties of studied HAs, which
was in conflict with out hypothesis. It was also demonstrated that there was no
relationship between biological activity and surface tension of studied HAs.
In overall results and from the point of view of biological and surface activity, the
most efficient modifier was 20% formic acid and the less efficient 20% propionic
acid.
31
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8 ABSTRACT
In this work, we tested the modified lignite humic acids for their remediation
capability of agricultural soils. Prior to the extraction of humic acids, the parental
raw lignite was modified by ten organic acids. The pre-treatment was aimed to
simulate similar processes that occur in rhizosphere, i.e. small-chain organic acids
induce the reconformation of soil organic matter thereby releasing biologically
active aggregates/molecules promoting the plant growth. In the first step, the
produced modified humic acids (MHA) were characterised for their physicochemical properties and molecular structure of by using elemental analysis (EA),
Fourier-transformed infrared (FTIR) spectroscopy, high-pressure size exclusion
chromatography (HPSEC), surface tension measurement and gas chromatographymass spectrometry (GC-MS) analysis. In the second step, the parameters of
biological activity were obtained from experiments oriented to both higher plants
and remediation of microbiological activity in used soil. The biological activity
towards higher plants was conducted on maize seeds and represented by total mass
and length increments of roots, root division and sugars and protein contents. The
influence on used soil was determined by laboratory soil incubation (CO 2
production) and soil water repellency measurement (contact angle). All results were
subjected to statistical analysis using Pearson´s correlation coefficient to find
relationship between physico-chemical properties and biological activity of studied
HAs samples. The results showed correlations between biological activity and
physico-chemical properties of humic acids. On the contrary, the surface properties
did not show any correlations with physical-chemical properties of studied HAs. The
most efficient modifier in terms of biological activity was 20% formic acid and the
less efficient was 20% propionic acid.
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